The long term goals of the proposed research are to develop reagents: (1) to identify a component(s) essential to chloride ion transport in airway epithelium, which is defective in cystic fibrosis (CF); and (2) to contribute to the basic understanding of the normal process of chloride ion transport at apical membrane sites. The first specific aim of this investigation is to prepare a series of novel photoaffinity analogues of 5-nitro-2-(3-phenylpropyl-amino)-benzoic acid (I), an extremely potent and effective inhibitor of chloride ion transport in the cortical thick ascending limb of the loop of Henle from rabbit. Compound I is an optimized version of the basic structure diphenylamine-2-carboxylate, an agent that blocks chloride ion transport at apical sites on bovine tracheal epithelia, the chosen model system for the proposed investigation. The synthetic photoaffinity (PAL) probes will be screened to identify ones that elicit irreversible chloride ion transport blockage upon irradiation. The second aim is to prepare radiolabeled versions of promising compounds identified by testing with the model membrane system. The third aim is to demonstrate covalent bond formation between the photoaffinity reagents(s) and chloride ion channel components. The importance of accomplishing the specific aims of this proposal is that by preparing and testing novel synthetic PAL reagents, new probes for chloride channel function will be made available for further study by other laboratories as well as our own. Future proposed investigations may be conducted using the PAL reagents described herein to elucidate the normal chloride transport process and the defective process in CF.